scholarly journals Nuclear Export of Glucocorticoid Receptor is Enhanced by c-Jun N-Terminal Kinase-Mediated Phosphorylation

2002 ◽  
Vol 16 (10) ◽  
pp. 2382-2392 ◽  
Author(s):  
M. Itoh ◽  
M. Adachi ◽  
H. Yasui ◽  
M. Takekawa ◽  
H. Tanaka ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Glucocorticoid Receptor ◽  
Nuclear Export ◽  
Biological Effect ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Uv Exposure ◽  
Green Fluorescent

Abstract The c-Jun N-terminal kinase (JNK) phosphorylates the glucocorticoid receptor (GR) and inhibits GR-mediated transcription. However, the biological effect of the GR phosphorylation remains unknown. Here we demonstrate that activated JNK phosphorylates human GR at Ser226 and enhances its nuclear export after withdrawal of a ligand for GR, dexamethasone. At 1 h after dexamethasone withdrawal, green fluorescent protein-GR molecules were mostly retained at the nucleus, whereas UV exposure enhanced its nuclear export, and approximately 30–40% of cells revealed distinct nuclear export. JNK overexpression alone mimics UV exposure and enhanced GR export accompanied by inhibition of GR-mediated transcription. However, mutation of the Ser226 JNK phosphorylation site in GR abrogated UV-mediated enhancement of GR nuclear export. Furthermore, overexpression of a dominant negative SEK1 mutant also abrogated the effects of UV exposure on GR export. Taken together, these findings suggest that JNK-mediated phosphorylation of the GR-Ser226 enhances GR nuclear export and may contribute to termination of GR-mediated transcription.

Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium

2000 ◽  
Vol 113 (12) ◽  
pp. 2253-2265 ◽  
Author(s):  
M. Dumontier ◽  
P. Hocht ◽  
U. Mintert ◽  
J. Faix
Keyword(s):  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Fluorescent Protein ◽  
Colony Growth ◽  
Dominant Negative ◽  
Related Protein ◽  
Wild Type ◽  
Equal Capacity ◽  
Green Fluorescent

The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.

scholarly journals Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants

2001 ◽  
Vol 15 (7) ◽  
pp. 1077-1092 ◽  
Author(s):  
Mayumi Nishi ◽  
Hiroshi Ogawa ◽  
Takao Ito ◽  
Ken-Ichi Matsuda ◽  
Mitsuhiro Kawata
Keyword(s):  
Green Fluorescent Protein ◽  
Glucocorticoid Receptor ◽  
Subcellular Localization ◽  
Mineralocorticoid Receptor ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Nuclear Accumulation ◽  
Accumulation Rates ◽  
Significant Difference ◽  
Green Fluorescent

Abstract Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are ligand-dependent transcription factors. Although it is generally accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial. We examined the intracellular trafficking of MR in living neurons and nonneural cells using a fusion protein of green fluorescent protein (GFP) and rat MR (GFP-MR). Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand. To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectively. In COS-1 cells, expressing no endogenous corticosteroid receptors, the YFP-MR chimera was accumulated in the nucleus faster than the CFP-GR chimera in the presence of 10−9m CORT, while there was no significant difference in the nuclear accumulation rates in the presence of 10−6m CORT. On the other hand, in primary cultured hippocampal neurons expressing endogenous receptors, the nuclear accumulation rates of the YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT. These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types.

Identification of a functional nuclear export signal in the green fluorescent protein asFP499

2006 ◽  
Vol 342 (4) ◽  
pp. 1178-1182 ◽  
Author(s):  
Huseyin Mustafa ◽  
Bernd Straßer ◽  
Sabine Rauth ◽  
Robert A. Irving ◽  
Kim L. Wark
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Green Fluorescent ◽  
Export Signal

scholarly journals RhoG GTPase Controls a Pathway That Independently Activates Rac1 and Cdc42Hs

1998 ◽  
Vol 9 (6) ◽  
pp. 1379-1394 ◽  
Author(s):  
Cécile Gauthier-Rouvière ◽  
Emmanuel Vignal ◽  
Mayya Mériane ◽  
Pierre Roux ◽  
Philippe Montcourier ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Local Concentration ◽  
Cell Periphery ◽  
Growth Factor Signaling ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Membrane Ruffling ◽  
Green Fluorescent

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

scholarly journals A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

1999 ◽  
Vol 144 (3) ◽  
pp. 389-401 ◽  
Author(s):  
Ed Hurt ◽  
Stefan Hannus ◽  
Birgit Schmelzl ◽  
Denise Lau ◽  
David Tollervey ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
In Vivo Assay ◽  
Green Fluorescent ◽  
Transport Factors

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

scholarly journals Kinetic Analysis of Smad Nucleocytoplasmic Shuttling Reveals a Mechanism for Transforming Growth Factor β-Dependent Nuclear Accumulation of Smads

2005 ◽  
Vol 25 (22) ◽  
pp. 9845-9858 ◽  
Author(s):  
Bernhard Schmierer ◽  
Caroline S. Hill
Keyword(s):  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Nuclear Export ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Shuttling ◽  
Green Fluorescent

ABSTRACT Upon transforming growth factor β (TGF-β) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-β-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-β-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-β-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.

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